This post was contributed by guest bloggers, Qi Li, a lecturer at Sichuan Normal University, Jieze Zhang, an intern in the Yang Lab, and Sheng Yang, a professor in synthetic biology from the Chinese Academy of Sciences.
The pCas/pTargetF system is one of the most popular genomic editing systems in E. coli. According to data from Addgene, MolecularCloud and our laboratory, there were 723 and 615 usage records in total for pCas and pTargetF respectively. However, some users pointed out that this system did not function properly in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF into the BL21(DE3)/pCas strain. Our lab at the Chinese Academy of Sciences optimized the pCas/pTargetF system and updated it into a pEcCas/pEcgRNA system. The work “A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli” was published in Acta Biochimica et Biophysica Sinica on March 25, 2021.
The original pCas/pTargetF system
Our original pCas/pTargetF system consists of one plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing, an arabinose inducible lambda-red gene, and an IPTG-inducible sgRNA sequence targeting the pMB1 replicon on pTargetF. In addition to the pMB1 replicon, pTargetF contains the sgRNA targeting the genomic site of interest. The donor template is supplied separately.
To use this system, you’d first introduce your sgRNA sequence into the pTargetF construct and then introduce both plasmids into the cell. The edit in the chromosome can now take place. After editing, pTargetF can be cured from the cell by inducing expression of the sgRNA that targets the pMB1 replicon, resulting in cutting of the plasmid.
At this point, subsequent introductions of pTargetF containing another sgRNA targeting another site of interest can be used to make other edits. Once you have all the edits you want, cure pCas9 by growing the cells at 37 °C.
The new pEcCas/pEcgRNA system
In the current study, we speculated that the gRNA on pCas that is specific to the pMB1 replicon of pTargetF may exhibit a more serious leaky expression in BL21(DE3), which we presume caused pTargetF to be cut by Cas9. This would result in the lack of recovered transformants. Our speculation motivated us to update the pCas/pTargetF system.
|Figure 1: The editing process begins with the addition of the sgRNA sequence in pEcgRNA. Next both plasmids are transformed in E. coli with the donor DNA for editing to take place. Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from Li et al., 2021.|
The updates involved the following changes. In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed the replicon of pCas into a non-temperature-sensitive replicon, and 3) added the sacB gene for counterselection to yield pEcCas. In pTargetF, we replaced the original 20 bp specific sequences on pTargetF with the ccdB gene containing BsaI restriction enzyme sites at its ends to generate pEcgRNA. The ccdB serves as a counterselection marker and will be replaced by the sgRNA targeting the genomic site of interest.
We compared the pCas/pTargetF system with the updated pEcCas/pEcgRNA system, and confirmed that indeed the gRNA-pMB1 had a slightly higher leaky expression in BL21(DE3) that resulted in a lower efficiency of transformation for pTargetF series plasmids. Fortunately, we were able to successfully delete genes of interest in BL21(DE3) using the newly generated pEcCas/pEcgRNA system.
Advantages of the pEcCas/pEcgRNA system
Compared with the original system, the pEcCas/pEcgRNA is superior in several ways:
- The pEcCas plasmid can be operated at 37 ℃ for faster growth of its E. coli host, and the addition of a sacB gene renders it beneficial for plasmid clearance.
- It is more convenient to update the spacer(20nt) on pEcgRNA plasmid as only two 24 nt oligos are required and the BsaI linearized pEcgRNA backbone can be prepared in large amounts and frozen for future use or directly ligated with the annealed 24 nt oligos to generate new pEcgRNA.
- pEcCas/pEcgRNA can be applied not only in coli K-12 strains, but also in E. coli B and W strains.
- The optimized plasmid curing method was much simpler and took only ~32 hours instead of ~60 hours.
We successfully tested the pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella citrea, with a total of 70 sites edited. We optimized the strategy for plasmid curing, providing users with a complete editing process. If you’ve given this system a try, we’d love to hear your feedback!
Thank you to our guest bloggers!
Qi Li obtained her Ph.D. from the Chinese Academy of Sciences (CAS) Center for Excellence in Molecular Plant Sciences in 2019. She joined Sichuan Normal University and became a lecturer in the College of Life Sciences that same year. Her research is focused on the escape mechanism of genome editing in microorganisms which could be applied to optimization of genome editing tools.
Jieze Zhang is an undergraduate student studying biochemistry at the University of Southern California. He is also an intern at the Yang Lab of the CAS Center for Excellence in Molecular Plant Sciences since December 2020. His research focuses on the efficiency of transposon-encoded CRISPR/Cas systems in bacteria, specifically on optimizing strains with low efficiency.
Sheng Yang obtained his Ph.D. from Shanghai Institute of Biochemistry, Chinese Academy of Sciences. He joined Shanghai Institutes for Biological Sciences in 2000 and became a professor in Synthetic Biology in 2006. The Yang lab's research is focused on developing microbial genome editing tools for faster construction or reprogramming of cells to understand the genetic and biochemical mechanisms behind their distinct phenotypes and to design a new generation of engineered cells for green chemistry and biomedical applications.
References and Resources
Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S (2015) Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Appl Environ Microbiol 81:2506–2514 . https://doi.org/10.1128/aem.04023-14
Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S (2021) A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochimica et Biophysica Sinica. https://doi.org/10.1093/abbs/gmab036
Additional resources on the Addgene blog:
- Browse CRISPR articles from our CRISPR topic overview
- Find other CRISPR methods for bacteria
- Find all articles about microbiology
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